The chromosomal translocations that are closely associated with cancer, most frequently hematopoietic malignancies, disrupt master regulatory genes. Myeloid Translocation Gene on chromosome 16 (MTG16, also known as Eto2) is disrupted by the t(16;21) chromosomal translocation in acute myeloid leukemia (AML), and this translocation is most commonly associated with therapy-related AML. In addition, deletion of MTG16 is observed in up to 40% of the most common form of breast cancer. ETO/MTG8 (RUNX1T1) is a closely related protein, which is disrupted by the t(8;21), which is one of the most frequent chromosomal translocations in AML. A third member of this gene family, Mtgrl, is not affected directly by translocations, but resides in a region of Chromosome 20 that is frequently deleted in myelo-proliferative disorders. MTG family members are nuclear proteins that appear to function as transcriptional co-repressors that link regulatory DNA binding proteins to histone modifying enzymes to repress and/or silence gene expression. To begin to understand the regulatory pathways that depend on Mtg16, we have deleted this gene from the germ line of mice. The Mtg16-null mice are mostly normal, but display mild hematopoietic defects. However, bone marrow transplantation assays revealed a dramatic defect in the function of hematopoietic stem cells and progenitor cells, as the bone marrow from Mtg16-null mice was unable to protect lethally irradiated recipient mice and poorly competed in competitive repopulation assays. Moreover, the Mtg16-null stem cells and progenitor cells were completely defective in spleen colony forming assays. Importantly for this proposal, we have been able to complement this defect in hematopoietic stem cell function by re- expression of Mtg16 via ex vivo retroviral transduction. Thus, we have a quick, robust method to assess Mtg16 action in HSCs in vivo. In addition, we have already created Mtgr1-deficient mice and found that these mice may also show signs of a stem cell defect in the colon upon stress. Finally, we have re- generated Mtg8-null chimeric mice, such that we can genetically dissect the action of the entire gene family by creating mice lacking Mtg16 and either Mtg8 or Mtgr1. Therefore, we will define the action of Mtg16 in the hematopoietic stem cell and determine the function of this key regulatory gene family in stem cell biology and cancer.